Spatial heterogeneity and peptide availability determine CTL killing efficiency in vivo

The rate at which a cytotoxic T lymphocyte (CTL) can survey for infected cells is a key ingredient of models of vertebrate immune responses to intracellular pathogens. Estimates have been obtained using in vivo cytotoxicity assays in which peptide-pulsed splenocytes are killed by CTL in the spleens of immunised mice. However the spleen is a heterogeneous environment and splenocytes comprise multiple cell types. Are some cell types intrinsically more susceptible to lysis than others? Quantitatively, what impacts are made by the spatial distribution of targets and effectors, and the level of peptide-MHC on the target cell surface? To address these questions we revisited the splenocyte killing assay, using CTL specific for an epitope of influenza virus. We found that at the cell population level T cell targets were killed more rapidly than B cells. Using modeling, quantitative imaging and in vitro killing assays we conclude that this difference in vivo likely reflects different migratory patterns of targets within the spleen and a heterogeneous distribution of CTL, with no detectable difference in the intrinsic susceptibilities of the two populations to lysis. Modeling of the stages involved in the detection and killing of peptide-pulsed targets in vitro revealed that peptide dose influenced the ability of CTL to form conjugates with targets but had no detectable effect on the probability that conjugation resulted in lysis, and that T cell targets took longer to lyse than B cells. We also infer that incomplete killing in vivo of cells pulsed with low doses of peptide may be due to a combination of heterogeneity in peptide uptake and the dissociation, but not internalisation, of peptide-MHC complexes. Our analyses demonstrate how population-averaged parameters in models of immune responses can be dissected to account for both spatial and cellular heterogeneity

Do Zebra Finch Parents Fail to Recognise Their Own Offspring?

Individual recognition systems require the sender to be individually distinctive and the receiver to be able to perceive differences between individuals and react accordingly. Many studies have demonstrated that acoustic signals of almost any species contain individualized information. However, fewer studies have tested experimentally if those signals are used for individual recognition by potential receivers. While laboratory studies using zebra finches have shown that fledglings recognize their parents by their “distance call”, mutual recognition using the same call type has not been demonstrated yet. In a laboratory study with zebra finches, we first quantified between-individual acoustic variation in distance calls of fledglings. In a second step, we tested recognition of fledgling calls by parents using playback experiments. With a discriminant function analysis, we show that individuals are highly distinctive and most measured parameters show very high potential to encode for individuality. The response pattern of zebra finch parents shows that they do react to calls of fledglings, however they do not distinguish between own and unfamiliar offspring, despite individual distinctiveness. This finding is interesting in light of the observation of a high percentage of misdirected feedings in our communal breeding aviaries. Our results demonstrate the importance of adopting a receiver's perspective and suggest that variation in fledgling contact calls might not be used in individual recognition of offspring

Interleukin-7 Influences FOXP3+CD4+ Regulatory T Cells Peripheral Homeostasis

Mechanisms governing peripheral CD4+ FOXP3+ regulatory T cells (Treg) survival and homeostasis are multiple suggesting tight and complex regulation of regulatory T cells homeostasis. Some specific factors, such as TGF-β, interleukin-2 (IL-2) and B7 costimulatory molecules have been identified as essentials for maintenance of the peripheral Treg compartment. Conversely, Treg dependency upon classical T cell homeostatic factors such as IL-7 is still unclear. In this work, we formally investigated the role of IL-7 in Treg homeostasis in vivo in murine models. We demonstrated that IL-7 availability regulated the size of peripheral Treg cell pool and thus paralleled the impact of IL-7 on conventional T cell pool. Moreover, we showed that IL-7 administration increased Treg cell numbers by inducing thymic-independent Treg peripheral expansion. Importantly the impact of IL-7 on Treg expansion was detected whether conventional T cells were present or absent as IL-7 directly participates to the peripheral expansion of Treg after adoptive transfer into lymphopenic hosts. Our results definitively identify IL-7 as a central factor contributing to Treg peripheral homeostasis, thus reassembling Treg to other T cell subsets in respect of their need for IL-7 for their peripheral maintenance

Tissue Specific Deletion of Inhibitor of Kappa B Kinase 2 with OX40-Cre Reveals the Unanticipated Expression from the OX40 Locus in Skin Epidermis

NF-κB signalling plays an essential role in T cell activation and generation of regulatory and memory populations in vivo. In the present study, we aimed to investigate the role of NF-κB signalling in post-activation T cells using tissue specific ablation of inhibitor of kappa-B kinase 2 expression, an important component of the inhibitor of kappa-B kinase complex in canonical NF-κB signalling. The OX40 antigen is expressed on activated T cells. Therefore, we used previously described mouse strain expressing Cre recombinase from the endogenous OX40 locus. Ablation of IKK2 expression using OX40Cre mice resulted in the development of an inflammatory response in the skin epidermis causing wide spread skin lesions. The inflammatory response was characterised by extensive leukocytic infiltrate in skin tissue, hyperplasia of draining lymph nodes and widespread activation in the T cell compartment. Surprisingly, disease development did not depend on T cells but was rather associated with an unanticipated expression of Cre in skin epidermis, and activation of the T cell compartment did not require Ikbk2 deletion in T cells. Employment of Cre reporter strains revealed extensive Cre activity in skin epidermis. Therefore, development of skin lesions was rather more likely explained by deletion of Ikbk2 in skin keratinocytes in OX40Cre mice

Skin pathology in <i>OX40^Cre Ikbk2^fx/fx</i> mice is associated with TNF production and increased apoptosis.

<p>(A) Confocal images of skins sections from 12–16 week old mice of the indicated strain were analysed for EYFP expression (green), stained with DAPI (blue) and for expression of TNF (red). White scale bar indicates 10 µm size. (B) Apoptosis was assessed in skin sections from <i>OX40^Cre Ikbk2^fx/fx and OX40^Cre Ikbk2^fx/wt</i> mice by TUNNEL assay and counterstained with eosin. Light microscopy was performed at 10× magnification.</p

Skin pathology in <i>OX40^Cre Ikbk2^fx/fx</i> mice is not T cell dependent.

<p>(A) Photograph shows skin pathology in a representative <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/fx</i> mouse (fx/fx) compared with a littermate <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/wt</i> control (fx/wt). White dotted line indicates the border between dermis and epidermis. (B) Images show skin sections from 12–16 week old mice of the indicated strain stained with H&E and taken at 10× magnification. (C) Confocal images of skins sections from 12–16 week old mice of the indicated mice stained for expression of CD45, cytokeratin 5 or cytokeratin 6 (red) and counter stained with DAPI. White scale bar indicates 10 µm size. (D) Graph shows progress of disease development in <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/fx</i> mice (blue, n = 18) as compared with Rag1 sufficient <i>OX40^Cre Ikbk2^fx/fx</i> mice (black lines, n = 9). (E) Bar chart shows mean time of disease progression to score 2 in <i>Rag1</i>^−/−<i>OX40^Cre Ikbk2^fx/fx</i> mice as compared with Rag1 sufficient <i>OX40^Cre Ikbk2^fx/fx</i> mice. Data are representative (A–C) of three independent experiments or are pooled (D–E) from three independent experiments.</p

<i>Cre</i> EYFP reporter expression in the skin of OX40^Cre strains.

<p>(A) Fluorescent stereomicroscope images of epidermal skin layer taken from the underside (×10 magnification). (B) Confocal images of skins sections from 12–16 week old mice of the indicated mice showing expression of EYFP and counter stained with DAPI. (C) EYFP expression in hair follicle of healthy <i>OX40^Cre Ikbk2^fx/wt</i> mice showing DAPI and EYFP separately and together. White scale bar indicates 10 µm size. Data are representative of five or more mice from three independent experiments.</p

Lymphoid hyperplasia and T cell activation in <i>OX40^Cre Ikbk2^fx/fx</i> mice.

<p>(A) Images show axillary and brachial lymph nodes (top four nodes) as compared with mesenteric chain and spleen from the indicated mouse strains, taken between 12–16 weeks of age. (B) Scatter charts show absolute numbers of total lymphocytes, CD4⁺ TCR^hi or CD8⁺ TCR^hi T cells in skin draining lymph nodes (dLN), mesenteric lymph nodes (mLN) or spleen (SPN) in <i>OX40^Cre Ikbk2^fx/fx</i> mice (fx/fx, n = 6) or control <i>OX40^Cre Ikbk2^fx/wt</i> mice (fx/wt, n = 4). (C) Density plots are of CD25 vs CD44 expression by CD4⁺ TCR^hi T cells (top row) and side scatter (SSC) vs CD44 by CD8⁺ TCR^hi T cells (bottom row) from dLN of the indicated mouse strains. Data are representative of six independent experiments.</p

<i>OX40^Cre Ikbk2^fx/fx</i> mice exhibit skin epidermis hyperplasia.

<p>(A) Images show skin sections from 12–16 wk old mice of the indicated strain stained with H&E and taken at 10× magnification. White dotted line indicates the border between dermis and epidermis. (B) Confocal images of skins sections from 12–16 week old mice of the indicated mice stained for expression of CD45, cytokeratin 5 or cytokeratin 6 (red) and counter stained with DAPI. White scale bar indicates 10 µm size. Data are representative of 3 independent experiments and at least two mice per strain experiment.</p